CN103598098B - Pachyrhizua angulatus tissue culturing fast seedling-cultivating method - Google Patents

Abstract

The present invention relates to Plant Tissue Breeding propagation technique field, a kind of Pachyrhizua angulatus tissue culturing fast seedling-cultivating method, comprise the steps: (1) pretreatment; (2) foundation of sterilizable material system; (3) Fiber differentiation; (4) propagation of bud; (5) culture of rootage; (6) acclimatization and transplants; Pachyrhizua angulatus tissue culturing fast seedling-cultivating method of the present invention, application method for tissue culture, effectively can keep the stable character of excellent female parent.Compare with propagation by layering with the cuttage of routine, tissue cultures with less excellent female parent, can breed large batch of seedling.At one time in section (60d), each female parent of cuttage can only breed an effective seedling, and the reproduction coefficient of tissue cultures is 20 ~ 30 times of cuttage.

Description

Rapid Seedling Raising Method of Pueraria kudzu Tissue Culture

technical field

The invention relates to the technical field of plant tissue culture and propagation, and relates to a rapid seedling raising method of kudzu tissue culture.

Background technique

Pueraria elobatavar. Thomsonii is a plant of the genus Pueraria in the family Leguminosae. There are 8 species and 2 varieties in my country, mainly distributed in the southwest, south-central to southeast, and rare in the north of the Yangtze River. The edible part of kudzu root is tuber, commonly known as " kudzu root ".

Pueraria lobata is a mountain plant recognized by the Ministry of Health of my country for both medicine and food. Puerarin is sweet, pungent, and flat in nature, and has the functions of relieving muscle and reducing fever, promoting body fluid, clearing rash, promoting yang and relieving diarrhea; the content of puerarin (isoflavones) is high, which can lower blood pressure, reduce myocardial oxygen consumption, slow down Heart rate, expansion of coronary vessels to improve the metabolism of normal and ischemic myocardium, improve cerebral circulation and peripheral circulation and so on. With the continuous development of modern medicine and food science and technology, the pharmacology, medicinal chemistry, clinical application, and food development and research of kudzu root have been continuously deepened, and its use value has attracted more and more people's attention, and it is known as "Asian ginseng". Authoritative organizations such as the World Food and Agriculture Organization predict that the global demand for kudzu root will reach 50 million tons per year (equivalent to 20 million mu).

In addition, the starch content of kudzu is high, and the starch content of some fine varieties can be as high as 25%. 7.5kg of fresh kudzu can produce 1kg of fuel ethanol. The ethanol productivity per unit area of kudzu is higher than that of corn, sweet potato and other food crops, and kudzu has high biological nitrogen fixation efficiency and is resistant to barrenness, which has the advantage of fully tapping the potential of the land. As a new non-grain biomass material, Pueraria kudzu avoids the bottleneck of "competing with the people for grain and land with grain", and has great development and utilization value.

Puerarialobata (wild) Ohwi'Dabashan' is a fine variety domesticated from wild kudzu varieties by the Dazhou Forestry Science and Technology Promotion Station. -PL-023-2009), has become the leading variety mainly promoted in the development of kudzu industry in Qinba Mountains, the development area is growing rapidly, and the demand for seedlings is large.

At present, the traditional method of layering and cutting propagation is the main method for the propagation of kudzu in Daba Mountain. The propagation coefficient is low, and the number of seedlings obtained is limited, which cannot meet the needs of current agricultural production. How to provide a large number of high-quality seedlings has become an urgent problem to be solved. question.

The rapid propagation of plant tissue culture is the most widely used field of contemporary agricultural biotechnology in production and produces the greatest economic benefits. Tissue culture rapid propagation of seedlings has its unique advantages. First of all, the reproduction coefficient is large, and it can be expanded and propagated at a speed thousands to millions of times faster than conventional methods. A large number of high-quality seedlings can be provided in time, so that a new variety can Meet the needs of production in a short period of time, and accelerate the promotion process of introduction and improved varieties; the propagation materials used are small and few; the propagation seedlings are neat and consistent, with high commerciality, and can maintain the excellent characteristics of the original varieties; annual industrialized seedling cultivation, production efficient. It is more important to use tissue culture for rapid propagation of plant varieties that have low reproductive coefficients and cannot be propagated by seeds (no seeds or seed-propagated offspring have species decline).

At present, although there have been patents and reports on the tissue culture seedling cultivation technology of several varieties of Pueraria genus Jingxi Pueraria, Pueraria Fenense, American Pueraria, Thai Pueraria and Pueraria mirifica, the tissue culture of different species, even the same species and different varieties, is different. There are also great differences in cultivation methods. Dabashan kudzu is a new variety, and there is no report on rapid seedling growth by tissue culture. Moreover, there are still some problems in kudzu tissue culture: it is difficult to ensure the genetic quality of the female parent by taking materials in the field; Therefore, it is an urgent task to expand the provenance production of Pueraria japonica in Daba Mountain and to solve the demand for seedlings in production to seek and explore the rapid propagation and seedling technology of kudzu tissue culture in Daba Mountain.

Contents of the invention

The object of the present invention is to provide a method for growing kudzu tissue culture seedlings, which uses tissue culture technology to increase the reproduction coefficient of kudzu and cultivate kudzu clones with stable genetic properties.

In order to achieve the above object, the technical solution adopted in the present invention is: a method for rapidly growing seedlings of kudzu tissue culture, comprising the steps of:

(1) Pretreatment Potted the roots of the excellent plants and placed them in the greenhouse. When the sprouted vines grow to 20cm, start to spray 1% carbendazim solution once a week for one and a half months, and start when the vines grow to 1m. Take materials;

(2) Establishment of aseptic material system Select kudzu vines of the same year, cut off the leaves and petioles, cut into 1.0-1.5cm long stems with axillary buds, wash the stems with buds under tap water for 30 minutes, and then use Soak in detergent powder for 10-30 minutes, scrub the stems with a soft brush, and then rinse them under running water for 1-2 hours. On an ultra-clean workbench, soak the washed stems in 75% alcohol for 15-20 seconds. Rinse with sterile water for 2 to 4 times, then soak with 0.1% mercuric chloride for 3 to 5 minutes, rinse with sterile water for 4 to 6 times, then treat with 2% sodium hypochlorite for 8 to 10 minutes, rinse with sterile water for 4 to 6 times, and use filter paper Blot dry the stem section with buds to obtain sterile material;

(3) Induction culture Inoculate the sterile bud stem section obtained in the above steps on 1/2MS medium, cultivate it for 10-15 days in the dark to reduce the occurrence of browning, and then transfer it to the bud induction medium to promote the growth of axillary buds. For germination and growth, the induction culture is alternately carried out according to the light and dark time ratio of 2:1;

(4) Proliferation of buds The buds are induced and cultured for 25 ± 2 days, the axillary buds germinate and grow to 1-1.5 cm, all of them are transferred to the proliferation medium for repeated proliferation, and after obtaining a large number of proliferation seedlings, they are transferred to rooting culture;

(5) rooting culture chooses 2～3cm buds from the multiplication culture to carry out rooting culture;

(6) Seedling hardening and transplanting Take out the tissue culture seedlings cultured by rooting from the cultivation room, move the culture bottle to the greenhouse for strong light closed bottle seedling training for 6-8 days, then open the mouth of the bottle, add PP ₃₃₃ and more The mixed solution of fungizolin prevents bacterial contamination and promotes strong seedlings. Place it for another 7-10 days. After the seedling hardening is completed, gently take out the test tube seedlings with tweezers, put them in a clean water basin, carefully wash off the agar at the roots, and then water them out. After the carbendazim treatment, transplant it into the nutrient soil. The upper layer of the nutrient soil is 2cm of river sand, and the lower layer is a mixture of 4cm of perlite, vermiculite and humus. After new leaves grow, it will be managed according to conventional nursery nursery measures.

Further: the kudzu root is selected from Dabashan kudzu root.

Further: the induction culture conditions of the step (2) are: temperature 24-26° C., light intensity 2500 lx, humidity 70%.

Further: the bud induction medium in the step (3) contains MS basal medium + 0.5-1.0 mg/L 6-BA + 0.1-0.3 mg/LIBA + 30 g/L sucrose + 7.0 g/L agar.

Further: the proliferation medium in the step (4) contains MS basal medium + 0.3-0.5 mg/L6-BA + 0-0.1 mg/LIBA + 30 g/L sucrose + 7.0 g/L agar.

Further: the rooting medium in the step (5) contains 1/2 MS basal medium + 0-0.1 mg/LIBA + 30 g/L sucrose + 7.0 g/L agar.

Further: the pH of the culture medium is 5.8-6.0.

Further: in the step (6) when the seedlings are hardened and transplanted, the air temperature is 20-25° C., and the humidity is 85%.

In the present invention, 1/2MS means that the macroelements in the MS medium are halved, and the others remain unchanged.

The beneficial technical effects of the present invention are: the fast seedling raising method of kudzu tissue culture of the present invention can effectively maintain the stable shape of an excellent female parent by applying the tissue culture method. Compared with conventional cutting and layering propagation, tissue culture can use fewer good female parents to reproduce a large number of seedlings. In the same period of time (60 days), each female parent of cuttings can only reproduce one effective seedling, and the propagation coefficient of tissue culture is 20 to 30 times that of cuttings.

detailed description

The present invention will be further described below in conjunction with specific implementation examples;

Implementation example one

A method for fast seedling cultivation of kudzu tissue culture, comprising the steps of:

(1) Pretreatment Potted roots of Dabashan Fengeyou strain were placed in a greenhouse, and when the sprouted vines grew to 20cm, sprayed 1% carbendazim solution once a week for one and a half months, and the vines grew to Start to collect materials at 1m;

(2) Establishment of the sterile material system Select kudzu vines that were born in the same year, cut off the leaves and petioles, cut into 1.0cm long stems with axillary buds, wash the stems with buds under tap water for 30 minutes, and then wash them with washing powder Soak the stems in liquid solution for 10 minutes, scrub the stems with a soft brush, and then rinse them under running water for 1 hour. On the ultra-clean workbench, soak the washed stems in 75% alcohol for 150 seconds, rinse them twice with sterile water, and then use Soak in 0.1% mercuric chloride for 3 minutes, rinse with sterile water for 4 times, then treat with 2% sodium hypochlorite for 8 minutes, rinse with sterile water for 4 times, dry the stem with buds with filter paper to obtain sterile materials;

(3) Induction culture Inoculate the sterile bud stem section obtained in the above steps onto 1/2 MS medium, cultivate it for 10 days in the dark to reduce the occurrence of browning, and then transfer it to the bud induction medium (the induction medium contains MS Basal medium+0.5mg/L6-BA+0.1mg/LIBA+sucrose 30g/L+agar 7.0g/L) to promote the germination and growth of axillary buds, and the induction culture was carried out alternately according to the light and dark time ratio of 2:1, and the culture temperature 24℃, light intensity 2500lx, humidity 70%;

(4) Proliferation of buds After bud induction culture for 23 days, the axillary buds germinate and grow to 1 cm, and all of them are transferred to the proliferation medium (the proliferation medium contains MS basal medium+0.3mg/L6-BA+0.1mg/LIBA+sucrose 30g/ L+agar 7.0g/L) carry out repeated multiplication culture, transfer to rooting culture after obtaining a large amount of multiplication seedlings;

(5) rooting culture choose 2cm shoots from the proliferation culture to carry out rooting culture (rooting medium contains 1/2MS basal medium+0.1mg/LIBA+sucrose 30g/L+agar 7.0g/L);

(6) Seedling hardening and transplanting The tissue cultured seedlings cultivated by rooting are taken out from the cultivation room, and the culture bottle is moved to the greenhouse to carry out strong light closed bottle training for 6 days, then open the bottle mouth, and then place it for 3 days. After the hardening is completed, use Gently take out the test-tube seedlings with tweezers, put them in a clear water basin, carefully wash off the agar at the root, then water them out, treat them with carbendazim, and transplant them into the nutrient soil. The upper layer of the nutrient soil is 2 cm of river sand, and the lower layer is 4 cm of perlite. , vermiculite and humus mixture, the air temperature is controlled at 20°C, and the humidity is 85%. After new leaves grow, manage them according to conventional nursery nursery measures.

All the culture media used above were adjusted with 1 mol/L HCl and NaOH to adjust the pH of the culture media to 5.8-6.0.

Implementation example two

A method for fast seedling cultivation of kudzu tissue culture, comprising the steps of:

(1) Pretreatment Potted roots of Dabashan Fengeyou strain were placed in a greenhouse, and when the sprouted vines grew to 20cm, sprayed 1% carbendazim solution once a week for one and a half months, and the vines grew to Start to collect materials at 1m;

(2) Establishment of the sterile material system Select kudzu vines that were born in the same year, cut off the leaves and petioles, cut into 1.3cm long stems with axillary buds, wash the stems with buds under tap water for 30 minutes, and then wash them with washing powder Soak the stems in liquid solution for 20 minutes, scrub the stems with a soft brush, and then rinse them under running water for 1.5 hours. On the ultra-clean workbench, soak the washed stems in alcohol with a mass concentration of 75% for 18 seconds, rinse them with sterile water for 3 times, and then Soak in 0.1% mercuric chloride for 4 minutes, rinse with sterile water for 5 times, then treat with 2% sodium hypochlorite for 9 minutes, rinse with sterile water for 5 times, dry the stem with buds with filter paper to obtain sterile materials;

(3) Induction culture Inoculate the sterile bud stem section obtained in the above steps onto 1/2 MS medium, cultivate for 13 days in the dark to reduce browning, and then transfer to bud induction medium (induction medium contains MS Basal medium+0.7mg/L6-BA+0.2mg/LIBA+sucrose 30g/L+agar 7.0g/L) to promote the germination and growth of axillary buds, and the induction culture was alternately carried out according to the light and dark time ratio of 2:1, and the culture temperature 25℃, light intensity 2500lx, humidity 70%;

(4) Proliferation of buds After bud induction culture for 25 days, the axillary buds germinate and grow to 1.2 cm, and all of them are transferred to the proliferation medium (the proliferation medium contains MS basal medium+0.4mg/L6-BA+0.1mg/LIBA+sucrose 30g /L+agar 7.0g/L) carry out repeated multiplication culture, transfer to rooting culture after obtaining a large amount of multiplication seedlings;

(5) rooting culture choose 2.5cm shoots from the proliferation culture to carry out rooting culture (rooting medium contains 1/2MS basal medium+0.1mg/LIBA+sucrose 30g/L+agar 7.0g/L);

(6) Seedling hardening and transplanting The tissue cultured seedlings cultivated by rooting are taken out from the cultivation room, and the culture bottle is moved to the greenhouse for strong light closed bottle training for 7 days, then the bottle mouth is opened, and then placed for 5 days. After hardening is completed, use Gently take out the test-tube seedlings with tweezers, put them in a clear water basin, carefully wash off the agar at the root, then water them out, treat them with carbendazim, and transplant them into the nutrient soil. The upper layer of the nutrient soil is 2 cm of river sand, and the lower layer is 4 cm of perlite. , vermiculite and humus mixture, the air temperature is controlled at 20°C, and the humidity is 85%. After new leaves grow, manage them according to conventional nursery nursery measures.

All the culture media used above were adjusted with 1 mol/L HCl and NaOH to adjust the pH of the culture media to 5.8-6.0.

Implementation example three

A method for fast seedling cultivation of kudzu tissue culture, comprising the steps of:

(1) Pretreatment Potted roots of Dabashan Fengeyou strain were placed in a greenhouse, and when the sprouted vines grew to 20cm, sprayed 1% carbendazim solution once a week for one and a half months, and the vines grew to Start to collect materials at 1m;

(2) Establishment of the sterile material system Select kudzu vines that were born in the same year, cut off the leaves and petioles, cut into 1.5cm long stems with axillary buds, wash the stems with buds under tap water for 30 minutes, and then wash them with washing powder Soak the stems with a soft brush for 30 minutes, wash the stems with a soft brush, and then rinse them under running water for 2 hours. On the ultra-clean workbench, soak the washed stems with 75% alcohol for 20 seconds, rinse them with sterile water for 4 times, and then use Soak in 0.1% mercuric chloride for 5 minutes, rinse with sterile water for 6 times, then treat with 2% sodium hypochlorite for 10 minutes, rinse with sterile water for 6 times, dry the stem with buds with filter paper to obtain sterile materials;

(3) Induction culture Inoculate the sterile bud stem section obtained in the above steps onto 1/2 MS medium, cultivate for 15 days in the dark to reduce browning, and then transfer to bud induction medium (induction medium contains MS Basal medium + 1.0mg/L6-BA + 0.3mg/LIBA + sucrose 30g/L + agar 7.0g/L) to promote the germination and growth of axillary buds, and the induction culture was carried out alternately according to the light and dark time ratio of 2:1, and the culture temperature 24℃, light intensity 2500lx, humidity 70%;

(4) Proliferation of buds After bud induction culture for 27 days, the axillary buds germinate and grow to 1.5cm, and they are all transferred to the proliferation medium (the proliferation medium contains MS basal medium+0.5mg/L6-BA+0.1mg/LIBA+sucrose 30g /L+agar 7.0g/L) carry out repeated multiplication culture, transfer to rooting culture after obtaining a large amount of multiplication seedlings;

(5) rooting culture choose 3cm shoots from the proliferation culture to carry out rooting culture (rooting medium contains 1/2MS basal medium+0.1mg/LIBA+sucrose 30g/L+agar 7.0g/L);

(6) Seedling hardening and transplanting The tissue cultured seedlings cultivated by rooting are taken out from the cultivation chamber, and the culture bottle is moved to the greenhouse to carry out strong light closed bottle seedling training for 8 days, the shading degree is 70%, then the bottle mouth is opened, and then placed for 7 days After hardening the seedlings, take out the test-tube seedlings gently with tweezers, put them in a clear water basin, carefully wash off the agar at the root, then water them out, treat them with carbendazim, and transplant them into the nutrient soil. The upper layer of the nutrient soil is 2 cm long. Sand, the lower layer is a mixture of 4cm perlite, vermiculite and humus, the air temperature is controlled at 20°C, and the humidity is 85%. After new leaves grow, it will be managed according to conventional nursery nursery measures.

All the culture media used above were adjusted with 1 mol/L HCl and NaOH to adjust the pH of the culture media to 5.8-6.0.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (4)

1. a fast seedling raising method of kudzu tissue culture, is characterized in that: comprise the steps: (1) Pretreatment Potted the roots of the excellent plants and placed them in the greenhouse. When the sprouted vines grow to 20cm, start to spray 1% carbendazim solution once a week for one and a half months, and start when the vines grow to 1m. Take materials; (2) Establishment of aseptic material system Select kudzu vines of the same year, cut off the leaves and petioles, cut into 1.0-1.5cm long stems with axillary buds, wash the stems with buds under tap water for 30 minutes, and then use Soak in detergent powder for 10-30 minutes, scrub the stems with a soft brush, and then rinse them under running water for 1-2 hours. On an ultra-clean workbench, soak the washed stems in 75% alcohol for 15-20 seconds. Rinse with sterile water for 2 to 4 times, then soak with 0.1% mercuric chloride for 3 to 5 minutes, rinse with sterile water for 4 to 6 times, then treat with 2% sodium hypochlorite for 8 to 10 minutes, rinse with sterile water for 4 to 6 times, use The filter paper blots dry the stem section with buds to obtain sterile material; (3) Induction culture Inoculate the sterile bud stem section obtained in the above steps on 1/2MS medium, cultivate it for 10-15 days in the dark to reduce the occurrence of browning, and then transfer it to the bud induction medium to promote the growth of axillary buds. For germination and growth, the induction culture is alternately carried out according to the light and dark time ratio of 2:1; (4) Proliferation of buds The buds are induced and cultured for 25 ± 2 days, the axillary buds germinate and grow to 1-1.5 cm, all of them are transferred to the proliferation medium for repeated proliferation, and after obtaining a large number of proliferation seedlings, they are transferred to rooting culture; (5) rooting culture chooses 2～3cm buds from the multiplication culture to carry out rooting culture; (6) Seedling hardening and transplanting Take out the tissue culture seedlings cultured by rooting from the cultivation room, move the culture bottle to the greenhouse for strong light closed bottle seedling training for 6-8 days, then open the mouth of the bottle, add PP ₃₃₃ and more The mixed solution of fungizolin prevents bacterial contamination and promotes strong seedlings. Place it for another 7-10 days. After hardening the seedlings, take out the test-tube seedlings gently with tweezers, put them in a clean water basin, carefully wash off the agar at the roots, and then remove them with After the carbendazim treatment, transplant it into the nutrient soil. The upper layer of the nutrient soil is 2cm river sand, and the lower layer is a mixture of 4cm perlite, vermiculite and humus. After new leaves grow out, manage them according to the conventional nursery nursery measures; The bud induction medium in the step (3) contains MS basal medium+0.5～1.0mg/L6-BA+0.1～0.3mg/LIBA+sucrose 30g/L+agar 7.0g/L; The proliferation medium in the step (4) contains MS basal medium+0.3～0.5mg/L6-BA+0～0.1mg/LIBA+sucrose 30g/L+agar 7.0g/L; The rooting medium in the step (5) contains 1/2MS basal medium+0～0.1mg/LIBA+sucrose 30g/L+agar 7.0g/L; The kudzu root is selected from Dabashan kudzu root. 2. The method for rapid seedling cultivation of kudzu tissue culture according to claim 1, characterized in that: the induction culture conditions of the step (3) are: temperature 24-26° C., light intensity 2500 lx, humidity 70%. 3. The method for rapid seedling cultivation of kudzu tissue culture according to any one of claim 1, characterized in that: the pH of the culture medium is 5.8-6.0. 4. The rapid seedling raising method of kudzu root tissue culture according to claim 1, characterized in that: the air temperature is 20-25° C. and the humidity is 85% when said step (6) hardening and transplanting.